FIGURE 5.

hnRNP K binds single-stranded LDLR probe containing CT-rich element of LDLR gene promoter. A, EMSA assays were carried out using 5′-biotin end-labeled single-stranded LDLR or c-myc DNA probes with LightShift® Chemiluminescent EMSA kit. Unlabeled single-stranded probes were used as specific competitors, and a DNA fragment from intron 1 of LDLR gene was used as a nonspecific competitor in 100-fold excess. To observe the specific shifted bands, recombinant hnRNP K protein was added to the reaction mixture. An antibody against hnRNP K was added into the DNA-protein reaction mixture to detect supershifted bands. B, EMSA assays were carried out to examine the ability of mutated R3D oligonucleotide to compete the K binding to the labeled probe. C, EMSA assays were carried out using 5′-biotin end-labeled single- or double-stranded LDLR promoter probes. Shifted and supershifted bands were detected by mixing recombinant hnRNP K protein or hnRNP K protein plus anti-hnRNP K antibody, respectively.